{"id":"doi:10.1002/anie.7714055","kind":"article","title":"Substrate-Dependent Crosslinking by the Cytochrome P450 From Aminopyruvatide Biosynthesis","authors":["Padhi C","Nguyen DT","Zhu L","Cha L","Wald JW","Mitchell DA","van der Donk WA"],"author_count":7,"year":2026,"published":"2026-09-15","venue":"Angewandte Chemie (International ed. in English)","cited_by":0,"doi":"10.1002/anie.7714055","arxiv_id":null,"nct_id":null,"source_url":"https://doi.org/10.1002/anie.7714055","archive_url":"https://space-kitty.com/papers/doi:10.1002/anie.7714055","updated":null,"pmid":"42745189","pmcid":"PMC13703928","openalex_id":null,"pdf_url":"https://europepmc.org/articles/PMC13703928/pdf","abstract":"Cytochrome P450s catalyze an array of reactions including crosslinking of aromatic side chains in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). ApyO is a cytochrome P450 that forms a C─C bond between two tyrosines in a YLY motif in the substrate ApyA, the precursor peptide of the RiPP aminopyruvatide. We utilized cell-free translation to generate ApyA variants and probe the substrate tolerance of ApyO. Through AlphaFold-based modelling and in vitro assays, we show that ApyO accepts the 10 C-terminal residues of ApyA and requires a conserved Arg/Lys in the substrate. Inspired by substrate sequences in orthologous biosynthetic gene clusters, we substituted one of the tyrosine residues with a tryptophan and observed that ApyO catalyzed formation of an N─C bond between the indole of Trp and Cε2 of Tyr. ApyO unexpectedly catalyzed formation of a C─O bond between the two tyrosine residues when we substituted the leucine residue in the YLY motif with tyrosine or tryptophan. A peptide containing a biaryl linkage and C-terminal aminopyruvate displayed sub-nanomolar inhibition of select proteases, with the aminopyruvate group critical for activity. Overall, this study demonstrates plasticity in the manner of macrocyclization catalyzed by the P450 ApyO.","abstract_license":"CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)","license":"CC-BY","is_open_access":true,"retracted":false,"peer_reviewed":null,"categories":[],"details":null,"provenance":"europepmc","attribution":"Abstract of \"Substrate-Dependent Crosslinking by the Cytochrome P450 From Aminopyruvatide Biosynthesis\" by Padhi C, Nguyen DT, Zhu L et al., https://doi.org/10.1002/anie.7714055, used under its Creative Commons Attribution license (CC-BY); markup removed, otherwise unmodified.","topics":[{"slug":"protein-design","name":"AI Protein Design & Structure Prediction","domain_slug":"biotech"}],"cited_in_dossiers":[]}